We have previously documented that interleukin 6 (IL-6) supports the growth of early murine plasma cell tumors (myelomas) and have found that the subsequent loss of IL-6-dependence represents a key step in the progression to a fully malignant cancer. We are now performing studies aimed at (1) advancing our understanding of the cellular and molecular pathways involved in interleukin-6 signal transduction and IL-6-mediated cancer cell growth and (2) identifying the steps/alterations that lead to growth factor-independent, malignant cell growth. In order to identify new cytoplasmic molecules that interact with the signal transducing component of the IL-6 receptor, gp130, we have expressed and immobilized a recombinant form of gp130 on agarose beads, and have analyzed the pattern and activity of cytoplasmic molecules that bind to this molecule. Isolation, sequencing and characterization of several gp130-binding molecules, one of which possesses kinase activity, is currently underway. In addition, we are in the process of identifying the gp130 binding sites and specific amino acids that are phosphorylated by these molecules. To assess the contribution of these phosphorylated amino acids to gp130 function, we have individually mutated these amino acids and are now in the process of expressing these gp130 mutants in IL- 3-dependent cell lines in which we have expressed IL-6 receptor components. We are also performing studies on the transcriptional regulation of junB, an immediate-early response gene that is upregulated by IL-6. Using antisense oligonucleotides to inhibit jun B expression, we have found that junB is essential for the proliferation and survival of murine myeloma cells. Using luciferase reporter constructs, we have conducted studies to identify IL-6 responsive elements that regulate the junB promotor. Deletional analysis identified a 200 base pair region that possesses the ability to upregulate the junB promotor by 75-fold. Sequencing the 200 bp region has revealed several known response elements, and a mutational analysis of this region has allowed us to tentatively identify the active element and the associated transcription factors that participate in junB regulation. To investigate the causes for the progression of Il-6 dependent plasmacytoma cells to growth factor independent proliferation, we have also initiated studies to determine which of the known steps in IL-6 signal transduction remain linked to IL- 6. Early results indicate that junB transcription is constitutively active in plasmacytoma cells that have become independent of IL-6, thus indicating that the alteration is "upstream" of junB.